We propose to continue our studies of membrane lipid asymmetry in natural membranes by following the action of phospholipid exchange proteins on inside-out and right side-out plasma membrane fragments isolated from mammalian cells grown in culture. Covalent labeling techniques will also be used to assess lipid asymmetry in these preparations. We propose to continue our studies of the interactions of artificially generated lipid vesicles with mammalian cells. Asymmetric vesicles will be prepared using appropriate donor lipids and phospholipid exchange proteins. Such vesicles will then be used to perturb the lipid asymmetry in the plasma membranes of living cells, using the existing technology of vesicle-cell fusion and vesicle-cell lipid transfer. The persistence of this new asymmetry in the cell surface will be investgated, and its possible effect on cell morphology and function will be explored. We plan to prepare fluorescent- and radioactively-labeled antibodies to purified phospholipid exchange proteins derived from several sources. These will be used to examine the distribution of such proteins within single cells by fluorescence microscopy and EM autoradiographic procedures.